We investigate the molecular mechanisms by which transmembrane proteins are sorted to different compartments of the endomembrane system such as endosomes, lysosomes and a group of cell-type-specific organelles known as lysosome-related organelles (e.g., melanosomes and platelet dense bodies). Sorting is mediated by recognition of signals present in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of membrane coats (e.g., clathrin coats). Among these adaptor proteins are the heterotetrameric AP-1, AP-2, AP-3 and AP-4 complexes, the monomeric GGA proteins, and the heteropentameric retromer complex. Proper sorting requires the function of additional components of the trafficking machinery that mediate vesicle tethering and fusion. Current work in our laboratory is aimed at elucidating the structure, regulation and physiological roles of coat proteins and vesicle tethering factors, and investigating human diseases that result from genetic defects (e.g., Hermansky-Pudlak syndrome;neurodegenerative and neurodevelopmental disorders) of these proteins. AP-1, AP-2, and AP-3 are clathrin-associated adaptor complexes that recognize two types of sorting signal referred to as tyrosine-based and dileucine-based. Previous studies showed that tyrosine-based signals bind to the mu1, mu2 and mu3 subunits, whereas dileucine-based signals bind to a combination (i.e., a hemicomplex) of two subunits, gamma-sigma1, alpha-sigma2 and delta-sigma3, from the corresponding AP complexes. Structure-based mutational analyses allowed us to pinpoint the exact location of the binding sites for dileucine-based sorting signals fitting the DEXXXLLI consensus motif on the AP-1, AP-2 and AP-3 complexes. The location and topography of the corresponding sites are similar, although the strength and amino acid requirements of different interactions depend on the exact sequence of the signal and the particular AP complex involved. We also demonstrated the occurrence of multiple AP-1 complexes resulting from combinatorial assembly of various gamma (i.e., gamma1 and gamma2) and sigma1 (i.e., sigma1A, sigma1B and sigma1C) subunit isoforms encoded by different genes. These AP-1 variants bind dileucine-based signals with marked preferences for certain sequences, implying that they are not functionally equivalent. Indeed, mutations in sigma1A and sigma1B have recently been shown to be the cause of two severe neurodevelopmental disorders known as the MEDNIK and Fried syndromes, respectively. Based on our work, we hypothesize that defects in these diseases are caused by abnormal sorting of specific cargo proteins having dileucine-based signals. The AP-4 complex is distinct from the other AP complexes in that it does not recognize canonical tyrosine-based and dileucine-based signals. We recently found that, instead, the mu4 subunit of AP-4 binds an YKFFE sequence from the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP). Biochemical and X-ray crystallographic analyses revealed that the properties of the APP sequence and the location of the binding site on mu4 are distinct from those of canonical tyrosine-based signals binding to the mu subunits of other AP complexes. Two APP-like proteins, APLP1 and APLP2, have related sequences that also interact with mu4. Disruption of the AP-4-APP interaction shifts the distribution of APP from endosomes to the trans-Golgi network and enhances gamma-secretase-catalyzed processing of APP to the pathogenic amyloid-beta peptide. These results demonstrate that APP and AP-4 engage in a novel type of signal-adaptor interaction that mediates transport of APP from the trans-Golgi network to endosomes, thereby reducing amyloidogenic processing of the protein. AP-4 should thus be considered a novel regulator of APP processing and trafficking, and a potential risk factor for Alzheimer's disease. In previous work, we found that a complex named GARP is a critical component of the molecular machinery that mediates retrograde transport of various cargo proteins, including sorting receptors, processing endopeptidases, fusogenic proteins and bacterial and plant toxins, from endosomes to the trans-Golgi network in mammalian cells. We showed that the molecular function of this complex is to promote tethering and fusion of endosome-derived transport carriers to the trans-Golgi network. Biochemical analyses showed that the human GARP complex comprises four subunits named Vps52, Vps53, Vps54 and Ang2. Interference with any of the GARP subunits blocks the delivery of many cargos to the trans-Golgi network, leading to global defects in lysosomal function, lipid traffic and autophagy. In collaboration with Aitor Hierro (CIC-bioGUNE, Bilbao, Spain), we solved the crystal structure of a C-terminal fragment from Vps54, the first atomic structure to be solved for any part of the GARP complex. This structure revealed that that GARP is related to other multisubunit tethering complexes such as the exocyst and COG. In addition, we found that a leucine-967 to glutamine substitution in Vps54 identified in the wobbler mouse, an animal model for amyotrophic lateral sclerosis (ALS), destabilizes the protein, leading to lower levels of the GARP complex in all tissues. The motor neuron degeneration that is characteristic of this mutant mouse is therefore due to decreased levels of GARP and the ensuing defects in retrograde transport.